Real-time local oxygen measurements for high resolution cellular imaging

Abstract

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O₂ (pO₂) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO₂ for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O₂-micro-sensor created by adsorbing O₂-sensitive and O₂-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O₂, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO₂ and micro-gradients of pO₂ without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO₂ and intracellular events can be imaged simultaneously. In addition, the response-time (t₀.₅ = 0.7 s, 0–160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO₂ while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨₘ); and (2) OxySplots measuring pO₂ during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca²⁺]ᵢ levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO₂ with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.